Chiral and optically active carboxylic acids are in demand for organic chemical synthesis as starting materials for a large number of pharmaceutical and crop protection active ingredients. Chiral carboxylic acids can be used for classical racemate resolution via diastereomeric salts. Of special interest are substituted and unsubstituted R-(−)- or S-(+)-mandelic acid. Enzymatic synthesis of said compounds is based on nitrilases.
Nitrilases are enzymes catalyzing the hydrolysis of nitriles into the corresponding carboxylic acids and ammonium ions (Faber, Biotransformations in Organic Chemistry, Springer Verlag, Berlin/Heidelberg, 1992, ISBN 3-540-55762-8). Nitrilases were initially discovered in plants (Thimann and Mahadevan (1964) Arch Biochem Biophys 105:133-141) and subsequently isolated from many microorganisms (Kobayashi and Shimizu (1994) FEMS Microbiology Letters 120:217-224) like those of the genera Pseudomonas, Nocardia, Arthrobacter, Fusarium, Rhodoccocus, Klebsiella, Aureobacterium, Alcaligenes, Rhodopseudomonas, Corynebacterium sp. strain KO-2-4, Acinetobacter, Bacillus, Variovorax, Brevibacterium, Caseobacter, Mycobacterium, and Candida. The nitrilases have varied substrate specificities but can be roughly clustered into three groups according to their specificity: nitrilases specific for aliphatic nitriles, nitrilases specific for aromatic nitriles, and nitrilases specific for arylacetonitriles (Kobayashi et al. (1993) Proc Natl Acad Sci USA 90:247-251; Kobayashi and Shimizu (1994) above mentioned; Lévy-Schil et al. (1995) Gene 161:15-20; Layh et al. (1998) J Mol Catal B: Enzymatic 5:467-474).
The enzymatic synthesis of chiral and achiral carboxylic acids and α-hydroxycarboxylic acids utilizing nitrilases is described in the art (e.g., Yamamoto et al. (1991) Appl Environ Microb 57:3028-3032; Faber, Biotransformations in Organic Chemistry, 2nd edn, Springer-Verlag, Berlin, 1995; Lévy-Schil et al. (1995) Gene 161:15-20; Cowan et al. (1998) Extremophiles 2: 207-216; WO 03/000840; WO 96/09403; WO 92/05275, EP-B 0 348 901; U.S. Pat. No. 5,283,193; EP-A-0 449 648, EP-B-0 473 328, EP-B-0 527 553, EP-B-0 332 379, U.S. Pat. No. 5,296,373, EP-A-0 610 048, EP-A-0 610 049, EP-A 0 666 320, WO97/32030). The disadvantages of these processes is that they often lead to products with only low optical purity and/or that they proceed with only low space-time yields. WO 01/34786 describes a modified nitrilase having an amino acid change of the cysteine residue at position 162. The nitrilase is used to hydrolyze 2-hydroxy-4-(methyl thio) butyronitrile. “Selectivity” within WO 01/34786 is defined as the ratio of the resulting products 2-hydroxy-4-(methyl thio) butyramide and 2-hydroxy-4-(methyl thio)butanoic acid. Nothing is said concerning selectivity of the nitrilase concerning different substrates.
Most nitrilases have a very narrow range of substrate acceptance, which made them only suitable for conversion of one or a few nitriles with acceptable efficiency. This is an obstacle for the development of a biocatalytic process for new products and leads to economically unattractive processes. Therefore, it is an objective to provide nitrilases with modified, preferably broader substrate acceptance.